Method for producing fibronectin and fibinogen composition using a polyalkylene glycol and glycine or b-alanine

ABSTRACT

A method for producing protein compositions comprising fibrinogen and fibronectin is disclosed, wherein a fibrinogen and fibronectin-containing starting solution is treated with a precipitating composition which comprises two different components that modify the solubility of fibrinogen and/or fibronectin, so that in a single-step precipitation a precipitate is formed which comprises fibrinogen and fibronectin, and the precipitate formed optionally is further treated by methods known per se.

[0001] The invention relates to a method for producing proteincompositions comprising fibronectin and fibrinogen and, optionally,further ingredients as well as to protein compositions obtainableaccording to this method.

[0002] Tissue adhesives based on fibrinogen (“fibrin adhesives”) havebeen known for a long time. They serve for a seamless orsuture-supporting connection of human or animal tissues or organ parts,for sealing wounds, haemostasis and assisting wound healing.

[0003] Their mode of action is based on an imitation of the final phaseof blood coagulation.

[0004] By the action of thrombin, (soluble) fibrinogen at first isconverted into fibrin monomers which aggregate spontaneously and form asticky mass, a so-called fibrin clot. Simultaneously, factor XIII (FXIII) present is activated by thrombin in the presence of calcium ionsto factor XIIIa. By the latter, the aggregated fibrin monomers and alsofibronectin possibly present are cross-linked to a high polymer by newpeptide bonds forming. By this cross-linking reaction, the strength ofthe clot formed is substantially increased. Generally, the clot adhereswell to wound and tissue surfaces, which i. a. leads to the adhesive andhaemostatic effect.

[0005] Therefore, fibrin adhesives frequently are used as two-componentadhesives which comprise the fibrinogen component together with athrombin solution which additionally contains calcium ions.

[0006] A particular advantage of a fibrin adhesive consists in that thelatter does not remain at its site of application as a foreign body, butis completely resorbed just as in natural wound healing, and is replacedby newly formed tissue. Various cells, such as, e.g., macrophages and,subsequently, fibroblasts migrate into the clot, lyse and resorb theclot material and form new tissue.

[0007] Although the complicated procedures of wound healing so far by nomeans have become completely clear, it is considered to be certain thatthe presence of fibronectin in the clot is of crucial importance for thegrowing in of cells and thus for wound healing.

[0008] A fibrin clot of optimum composition threrefore should alsocomprise a content of fibronectin in addition to its main component,fibrinogen.

[0009] Although the mode of action of fibrin adhesives substantiallycorresponds to the natural processes of blood coagulation, for asufficient efficacy (adhesive strength, haemostatic effect), asubstantially higher concentration of the active components (inparticular, of fibrinogen) is necessary than is present in blood, thefibrinogen concentration of human blood amounting to approximately 2.5 -3 mg/ml.

[0010] It has been reported that by means of PEG precipitation afibrinogen solution could be obtained with which a satisfactory adhesivestrength could be attained already at fibrinogen concentrations of below30 mg/ml in an artificial test system (WO 92/13495), yet with this, formany purposes of tissue adhesion a high density of the fibrin network(and the latter is substantially only attainable by high fibrinogenconcentrations in the tissue adhesive) cannot be ensured,

[0011] To ensure an optimum efficacy, therefore, the fibrinogen contentin a fibrin adhesive should be at least 70 mg/ml. The production of suchconcentrated ready-to-use fibrinogen or fibrin adhesive solutions,respectively, does, however, involve some difficulties:

[0012] Since the ready-to-use solutions are not storage-stable overlonger periods of time, they have to be prepared upon demand either byreconstitution from lyophilized preparations or by thawing ofliquid-deep frozen solutions.

[0013] Because of the relatively poor solubility of fibrinogen and thesimultaneously required high fibrinogen concentration in an effectivefibrin adhesive, in general this is still more cumbersome andtime-consuming than desired by users thereof, despite diverse proposalsfor improvements. It is understandable that particularly in the field ofemergency surgery, a particularly rapid and simple availability of afibrin adhesive is required.

[0014] Moreover, concentrated fibrin adhesive solutions generally arehighly viscous due to their high fibrinogen concentrations. A relativelylow viscosity, however, is desirable not only for easier handling, butalso for specific modes of application of a fibrin adhesive, e.g. whenapplying it by means of spray devices (such as, e. g., Duploject® withthe associated spray set), or by means of a catheter.

[0015] Both requirements, i.e. rapid availability and low viscosity ofthe ready-to-use fibrin adhesive solutions, are even more difficult tomeet if preparation thereof (dissolution or thawing, respectively) is tobe effected without further auxiliary means, such as heating and/orstirring equipment, at room temperature, and if the fibrin adhesivepreparations additionally contain high-molecular substances, inparticular fibronectin. For also fibronectin—particularly in combinationwith fibrinogen—is relatively difficult to dissolve and generally leadsto an even poorer solubility and an increased viscosity of fibrinadhesives.

[0016] Methods for producing fibrinogen-containing preparations whichcan be used as tissue adhesives comprise i. a. their production fromcryoprecipitate, optionally with further washing and precipitation stepswith ethanol, ammonium sulphate, polyethylene glycol, glycine orβ-alanine, and their production from plasma within the scope of theknown plasma fractionation methods, respectively (cf., e.g., “Methods ofplasma protein fractionation”, 1980, ed.: Curling, Academic Press, pp.3-15, 33-36 and 57-74, or Blomb{overscore (a)}ck B. and M.,“purification of human and bovine fibrinogen”, Arkiv Kemi 10, 1959, p.415 f.).

[0017] In the prior art, also various suggestions have been made toreduce the viscosity of highly concentrated fibrinogen solutions. Thus,e.g., the addition of solubilizers, such as substances containing ureaor guanidine residue, e.g. arginine (cf. DE 3203775-Al), or the additionof unphysiologically high salt concentrations has been known. However,it has been shown that such tissue adhesives have cytotoxic andproliferation-inhibiting properties, respectively (Redl et al., Med.Welt 36, 1985, pp. 769-776)

[0018] According to EP 0 804 933, the addition of substances improvingthe solubility of fibrinogen has been suggested. Such substances are,e.g., vitamins, aromatic compounds, such as compounds derived frombenzene or phenol, or those derived from heterocyclic compounds, such aspiperidine, pyridine or pyrimidine.

[0019] Thus, it is an object of the present invention to overcome thedrawbacks of known preparations, and to further improve the knownpreparations, respectively, and to provide protein compositionscomprising a high fibrinogen content and an easily adjustable ratio offibrinogen to fibronectin as well as, optionally, further ingredients,which are, e.g., suitable for an improved preparation of ready-to-usetissue adhesives, while particularly maintaining properties, such as agood cell compatibility, or the formation of a physiological fibrinstructure after mixing with a thrombin solution. At the same time, alsothe viscosity properties of such protein compositions or pharmaceuticalpreparations are to be improved

[0020] A further object of the present invention is to provide asimplified and more rapid method of producing such protein compositions.In particular, it shall also be possible to easily carry out the methodon an industrial scale.

[0021] According to the invention, these objects are achieved by amethod for producing protein compositions, which comprise fibrinogen andfibronectin, which method is characterized in that a starting solutioncomprising fibrinogen and fibronectin is treated with a precipitatingcomposition which comprises two different components that modify thesolubility of fibrinogen and/or fibronectin, so that in a single-stepprecipitation a fibrinogen and fibronectin containing precipitate isformed, and the formed precipitate optionally is further treated bymethods known per se. This method is characterized in that an efficientand protein-preserving preparation giving high yields can be producedwith the precipitating composition by a single-step precipitation.

[0022] Following the single-step precipitation, the precipitate obtainedmay be further processed by methods known per se, preferably into apharmaceutical preparation, in particular a tissue adhesive (fibrinadhesive).

[0023] By a solubility-modifying component, a substance is understoodwhich has a precipitating effect or a solubility-improving effect on atleast one of the proteins, fibrinogen or fibronectin, under the givenother conditions, such as temperature, pH, ionic strength, etc..

[0024] Quite surprisingly, according to the invention it has been foundthat using two solubility-modifying components which are different (fromeach other) in cooperation allows for a recovery of fibrinogen andfibronectin in the precipitate in high yields, wherein the single-stepprecipitation of the invention quite amazingly has been found to besubstantially less protein-damaging than the individual precipitation ofthe respective substances.

[0025] Moreover, it has also been shown that a composition precipitatedaccording to the invention and comprising fibrinogen and fibronectin hasenormously improved properties also with a view to its practicalapplication, in particular as regards its viscosity and reconstitution,whereby an easier application of these preparations is made possible.

[0026] As the starting materials for preparing the starting solution, inprinciple all those can be used which so far have been used or arepossible, respectively, in the prior art for preparing such proteincompositions.

[0027] As the starting material, preferably, plasma, in particular humanplasma, or a fibrinogen and fibronectin-comprising plasma fraction,preferably a fraction derived from cryoprecipitate, is used forpreparing the starting solution or is used directly as startingsolution, respectively.

[0028] Other fibrinogen and fibronectin-comprising starting materials orstarting solutions, respectively, such as, e.g., cell culturesupernatants, may, however, also be employed according to the invention.

[0029] The solubility-modifying components are chosen such that in asingle-step precipitation a precipitate is formed which containsfibrinogen and fibronectin in a desired and pre-determined ratio ofamounts.

[0030] According to the invention, the components contained in theprecipitating composition are chosen such that, under the givenconditions, they differ from each other in terms of theirsolubility-modifying, i.e. solubility-improving or precipitating, actionon fibrinogen and fibronectin, respectively. One component may, forinstance, have a precipitating effect substantially on merely one of thetwo proteins, i.e. on fibrinogen or on fibronectin.

[0031] It is, however, also possible that one of the components has aprecipitating action on both proteins, whereas the other component has aprecipitating action on merely one of the two proteins, or evenincreases the solubility of the other protein, respectively, i.e. doesnot act as a precipitating agent but as a solubilizer.

[0032] In particular, the solubility-modifying component is a substanceselected from the group of alcohol, in particular alcohol having up tofour carbon atoms, inorganic salt, organic salt, polyol, in particularpolyalkylene glycol, polyether and amino acid. Also ethers, ketones andcyclic, heterocyclic or polycyclic organic compounds may be used. As thesolubility-modifying components, particularly ethanol, polyethyleneglycol, ammonium or alkali salts, respectively, such as ammonium sulfateor alkali sulfate, or the amino acids glycine and (β-) alanine arepreferably used.

[0033] Among the organic polymers, there are particularly the linearpolymers, in particular those having an average molecular weightapproximately between 200 and 20,000, e.g. polyalkylene glycols.Polyethylene glycol has proven particularly suitable. These non-toxic,water-soluble synthetic polymers, in particular those having a molecularweight of from 400 to 10,000, preferably around 4,000, are preferablyused in the precipitation because of their preserving effect onfibrinogen and fibronectin. In addition to the particularly gentle andpreserving effect of the components to be used in the precipitatingcomposition, also their safety as regards their use on humans is takeninto consideration when selecting them.

[0034] As a pair of components usable according to the invention, forinstance, polyethylene glycol (PEG), in particular PEG 4000 which has aprecipitating action both on fibrinogen and on fibronectin, and, as thesecond component, glycine is usable, which in the presence of a furthercomponent, e.g. polyethylene glycol, surprisingly not only preferablyprecipitates fibrinogen as compared to fibronectin, but even acts as asolubilizer for fibronectin. Glycine in a concentration range of morethan 0.6 M, in which so far glycine has only been known as a proteinprecipitating agent, has a particularly good, fibronectin-solubilityincreasing effect.

[0035] Of course, the present invention is not limited to combinationsof PEG and glycine, since the average skilled artisan will easily beable to find also other components or component pairs, respectively,suitable according to the invention, by means of simple systematictests.

[0036] To carry out the method of the invention, the skilled artisanmay, e.g. find suitable components or component pairs, respectively,according to the following test scheme. Under defined conditions, suchas temperature, pH, ionic strength etc. and under stirring, aliquots ofa starting solution prepared by common methods are admixed withprecipitating compositions containing a first solubility-modifyingcomponent, e.g. an amino acid, and a second component differenttherefrom, e.g. PEG 4000, ethanol or ammonium sulfate. Precipitationseries are carried out in which the concentration of one of thecomponents always is kept constant, and the other one is varied. Theprecipitates formed are centrifuged off, and the total protein contentas well as the relative contents of fibrinogen (Fbg), fibronectin (Fn)and albumin (Alb) are determined, e.g. by means of SDS PAGE of thenon-reduced and reduced samples, staining with Coomassie Blue anddensitometric evaluation.

[0037] Those components which lead to the desired fibrinogen andfibronectin-containing protein composition are then chosen for theprecipitating composition according to the invention. Optimization ofthe respective component system will be effected by means of parameterseasy to check, such as yield, relative content of fibrinogen andfibronectin, and viscosity, respectively, of the obtained preparation ata pre-determined fibrinogen-content (e.g. 70 or 100 mg of fibrinogen perml).

[0038] In a preferred embodiment, the precipitating compositioncomprises polyalkylene glycol which is admixed to the starting solution,in particular to a final concentration of from 1 to 12% w/v, preferablyfrom 4 to 8% w/v.

[0039] In a further preferred embodiment, the precipitating compositioncomprises one or more amino acids admixed to the starting solution, inparticular to a final concentration of from 0.5 to 3 M, preferably 0.75to 2 M, in particular 0. 75 to 1.5 M.

[0040] In another preferred embodiment, an organic or inorganic salt,e.g. ammonium sulfate, is contained at a concentration of up to 20% w/v,preferably between 2 to 20% w/v, most preferred between 5 to 15% w/v.

[0041] The precipitating composition preferably is admixed in liquidform.

[0042] Also other ingredients of the starting material can beprecipitated by means of the precipitating composition, such as inparticular factor XIII. Also the ability of the solubility-modifyingcomposition for co-precipitating such further substances may influencethe final choice of the individual components in the precipitatingcomposition.

[0043] According to a preferred embodiment it is attempted to keep theplasminogen in the precipitated protein composition or in thepharmaceutical preparation, respectively, rather low. Accordingly,preferably a precipitating composition is used in which plasminogenlargely remains in the supernatant, which means that in the precipitateobtained, plasminogen is depleted relative to fibrinogen and/orfibronectin, as compared to the starting material.

[0044] According to a further preferred embodiment, further ingredients,in particular fibrinolysis inhibitors, factor XIII, antibiotics, growthfactors, pain-alleviating substances, anti-inflammatory agents ormixtures thereof may be added to the protein composition. Such additionsare possible before, during, and also after the precipitation step andpreferably are effected after the precipitation step.

[0045] In the course of further processing, in particular in the finalformulation, the protein composition according to the invention or thepharmaceutical preparation according to the invention preferably isdeep-frozen or lyophilized, particularly if an improved storagestability is to be achieved. The ready-to-use pharmaceutical preparationaccording to the present invention preferably is provided in liquidform.

[0046] In particular, the method according to the invention has theadvantage that well-defined protein compositions are obtainable directlyin a simple manner, whereas hitherto used methods of proteinfractionation, in particular of plasma fractionation, always have aimedat obtaining individual proteins as pure as possible and, optionally, atmixing them together again afterwards to give the desired mixture.

[0047] By the simplicity and brevity of-the method according to theinvention, also the danger of microbial contamination and the formationof pyrogens during production are minimized. Moreover, sensitive plasmaproteins, such as, e.g., fibrinogen or fibronectin, are preserved andlargely protected from denaturing, and this is probably why thepreparations according to the invention have improved properties.

[0048] For surprisingly it has been shown that the protein compositionsor pharmaceutical preparations, respectively, obtainable by thesingle-step combination precipitation of the invention, at similarlyhigh fibrinogen concentrations have an approximately 25%, preferablyeven 30%, most preferred 40%, lower viscosity than comparablepreparations, e.g. fibrin adhesives obtained by the classical productionmethods, in particular by precipitation with one agent only.

[0049] By comparable preparations, preparations are understood whichhave similar contents of fibrinogen, fibronectin and, optionally,further ingredients, and a comparable degree of purity, which exhibit asimilarly good cell compatibility and which form clots of physiologicalfibrin structure after having been mixed with a thrombin solution.

[0050] In a particularly preferred embodiment, the protein compositionand the ready-to-use pharmaceutical preparation, respectively,substantially do not contain any substances improving the solubility ofthe fibrinogen, and no such substances are admixed, respectively.

[0051] Surprisingly it has been shown that liquid compositions accordingto the invention have a markedly lower viscosity as compared tocomparable known preparations, whereby particularly handling and use offibrin adhesives is facilitated. Particularly at room temperature, thelyophilized compositions according to the invention can be reconstitutedmore easily and more rapidly than comparable preparations.

[0052] Particularly when carrying out the method of the invention on alarge technical scale which—and this is a further advantage of thepresent invention—can be applied on a large scale very efficiently andin a cost-saving manner, it is preferred to admix the precipitatingcomposition in liquid form. This may be a solution or a suspension.

[0053] It has also proven suitable that the precipitate obtained afterthe single-step precipitation is washed at least once with a washingbuffer. In addition to a suitable buffer system, the washing bufferpreferably contains tranexamic acid, lysine, ε-aminocaproic acid,detergents, or mixtures of these substances. Also fibrinolysisinhibitors or protease inhibitors, respectively, of plasmatic, animal orplant origin, or inhibitors produced by genetical engineering orsynthetically, respectively, may be contained. Such substances may beadmixed separately, or they may already be contained in the startingmaterial.

[0054] A preferred protease inhibitor is, e.g., aprotinin, in particularaprotinin prepared by genetical engineering. A further preferredfibrinolysis inhibitor .is tranexamic acid(trans-4-(aminomethyl)-cyclohexane carboxylic acid, t-AMCHA).

[0055] The protein precipitate obtained may be used as such, or it maybe further processed to a further protein composition or to apharmaceutical preparation, respectively, by methods known per se. Suchmethods for processing include, e.g., diverse purification steps, suchas, e.g., treatment (washing) with a buffer solution in the cold, orformulation as a pharmaceutical preparation, respectively.

[0056] In particular, such final compositions or preparations,respectively, of the invention are suitable for tissue adhesion,haemostasis, and aiding or promoting wound healing.

[0057] Plasma which preferably is used as the starting material for thepresent invention, preferably is sufficiently pre-selected, e.g.according to the method of WO 96/35437, so that virus contamination cannearly be excluded. Preferably, only such a starting material is usedwhich has been checked for an absence of pathogens, in particularviruses and/or prions.

[0058] In a further preferred embodiment, at least one step forinactivating or depleting possibly present pathogens is provided.

[0059] To inactivate pathogens, preferably a tenside and/or heattreatment (dry or vapor treatment) is carried out, e.g. a heat treatmentin the solid state, in particular a vapor treatment according to EP-0159 311, or EP-0 519 901, or EP-0 674 531.

[0060] Further treatments for pathogen inactivation also comprisetreatment with chemical or chemical/physical methods, e.g. withchaotropic substances according to WO94/13329, DE 4434538 or EP-0131740(solvent), or photoinactivation.

[0061] Also the depletion by means of filtration, in particularultrafiltration, preferably in the presence of virus-binding agents,such as, e.g. Aerosil, is a preferred method for depleting viruseswithin the scope of the present invention.

[0062] According to the invention, inactivation by means of a heattreatment, a solvent treatment, a detergent treatment, or a combination(simultaneous or sequential) of these treatments, as well as,optionally, a filtration, are particularly preferred.

[0063] Inactivation may be effected before or after the precipitation.Preferably, two independent inactivations are carried out, e.g., onetreatment before the single-step precipitation, and a second treatmentthereafter.

[0064] In a further preferred embodiment, the protein composition ischaracterized in that factor XIII is contained. Preferably, factor XIIIis admixed, and the factor XIII content is, e.g., at least 80 units perg of fibrinogen, preferably at least 100 U/g of fibrinogen. In case alsothe fibrin-cross-linking reaction-inhibiting substances, such as, e.g.,antibiotics, are present in the preparation, the factor XIII contentpreferably is increased to at least 500 U/g of fibrinogen. Preferably,factor XIII is admixed as a purified, separately virus-inactivatedproduct (particularly preferably a factor XIII preparation producedaccording to EP 0 637 451).

[0065] In a further preferred embodiment; the protein composition ischaracterized by a low plasminogen content. The content of plasminogenpreferably is 1.6 mg/g of fibrinogen at the most, more preferred lessthan 0.8 mg/g of fibrinogen, most preferred less than 0.3 mg/g offibrinogen.

[0066] The protein composition according to the invention advantageouslycomprises fibronectin and fibrinogen at a ratio of from 0.02 to 0.5,preferably from 0.02 to 0.25, also preferred from 0.02 to 0.2, morepreferred from 0.04 to 0.16, most preferred around 0.1 (0.05 to 0.15),i.e., in contrast to highly pure fibrinogen preparations (WO 94/20524),it still contains substantial portions of fibronectin.

[0067] The protein composition may further comprise active ingredients,such as antibiotics, growth factors, pain alleviating substances orcombinations thereof.

[0068] In a preferred embodiment, the protein composition according tothe invention is characterized in that

[0069] a) as a solution, it contains at least 70 mg of fibrinogen/ml orcan be reconstituted or liquefied, respectively, to such a solution,

[0070] b) at 20° C., it has a viscosity of 350 cSt at the most,preferably at an osmolarity of less than 500 mOsm, most preferred ofless than 400 mOsm,

[0071] c) after mixing with a thrombin-CaCl₂ solution, it forms anon-transparent clot of physiological fibrin structure, and

[0072] d) it does not contain an addition of a solubility-improvingsubstance with a benzene, pyridine, piperidine, pyrimidine, morpholine,pyrrole, imidazole, pyrazole, furan, thiazole or purin-containing group.

[0073] In a further preferred embodiment, the protein compositionaccording to the invention is characterized in that

[0074] a) as a solution it contains at least 70 mg of fibrinogen/ml, orcan be reconstituted or liquefied, respectively, to such a solution,

[0075] b) at 20° C., it has a viscosity of 150 cSt at the most,preferably at an osmolarity of less than 500 mOsm, most preferred ofless than 400 mOsm,

[0076] c) after mixing with a thrombin-CaCl₂ solution, it forms anon-transparent clot of physiological fibrin structure, and

[0077] d) it contains one or several solubilizing substances with abenzene, pyridine, piperidine, pyrimidine, morpholine, pyrrole,imidazole, pyrazole, furan, thiazole or purine-containing group at aconcentration of a total of 150 mM at the most.

[0078] The inventive protein compositions or pharmaceuticalpreparations, respectively, are versatile in terms of use. inparticular, the preparations according to the invention are used fortissue adhesion, haemostasis and/or wound healing.

[0079] In a further preferred embodiment, the inventive proteincompositions or preparations, respectively, can also be used to producea fibrin-based biomatrix. To this end, the solution of a proteincomposition or pharmaceutical preparation, respectively, of theinvention is admixed with an enzyme suitable for converting fibrinogeninto fibrin, preferably with thrombin, and the fibrin formed is usedeither in its fresh state or after lyophilisation and re-moistening, asthe carrier material for growing cells, or as a so-called biomatrix(cf., e.g., WO 99/15209)

[0080] The biomatrix of the invention may be present in various forms,e.g. as a sponge, a film, as micro-beads or as flakes.

[0081] The fibrin-based biomatrix is particularly suitable for growingcells, in particular human cells. Keratinocytes, fibroblasts,chondrocytes are to be mentioned as an example of cells capable of beingcultured or grown by means of this fibrin matrix. Such a biomatrix isalso suitable for dressing wounds or as tissue substitute, respectively,particularly as a skin substitute.

[0082] The invention will now be explained in more detail by way of thefollowing examples and the drawing figures, to which, however, it shallnot he restricted.

[0083]FIG. 1 shows the dependence of the ratio of fibronectin tofibrinogen (Fn/Fbg) in the precipitate on the PEG concentration in theprecipitation mixture (glycine concentration: 1 M)

[0084]FIG. 2 shows the dependence of the ratio of fibronectin tofibrinogen (Fn/Fbg) in the precipitate on the glycine concentration inthe precipitation mixture (PEG concentration: 6.5% w/v)

[0085]FIG. 3 shows the dependence of the ratio of fibronectin tofibrinogen (Fn/Fbg) in the precipitate on the β-alanine concentration inthe precipitation mixture (ethanol concentration: 2% v/v)

[0086]FIG. 4 shows the dependence of the ratio of fibronectin tofibrinogen (Fn/Fbg) in the precipitate on the ammonium sulfateconcentration in the precipitation mixture (β-alanine concentration: 1M)

[0087]FIG. 5 shows the dependence of the ratio of fibronectin tofibrinogen (Fn/Fbg) in the precipitate on the β-alanine concentration inthe precipitation mixture (ammonium sulfate concentration: 5% w/v)

EXAMPLES Example 1

[0088] Human plasma cryoprecipitate prepared according to methods knownper se was dissolved with the 4-fold amount of a buffer solutioncontaining 20 mM sodium citrate, 120 mM sodium chloride, 5 mM tranexamicacid as well as 1200 IU heparin/l, the pH was adjusted to 7.3, and itwas filtered to clarification. Aliquots of this solution were admixedwith solutions containing glycine and polyethylene glycol 4000 (PEG4000) under stirring at room temperature, so that in each case a finalconcentration of 1 M glycine, yet different final concentrations of PEG4000 ranging from 1 to 10% (w/v) were obtained.

[0089] The precipitates formed were centrifuged off, and the relativecontent of fibrinogen (Fbg), fibronectin (Fn) and albumin (Alb) wasdetermined by means of SDS-PAGE of the non-reduced and reduced samples,staining with Coomassie Blue and densitometric evaluation (cf. also EP 0345 246, Examples 1-37)

[0090] The results have been summarized in Table 1; the ratio offibronectin : fibrinogen in dependence on the PEG concentration is alsographically represented in FIG. 1. TABLE 1 Dependence of the proteincomposition of the precipitate on the PEG content in the precipitationmixture (glycine concentration: 1 M) PEG Fibrinogen Fibronectin AlbuminFibronectin: % (w/v) % Protein % Protein % Protein Fibrinogen 1 84 3 50.04 2 83 4 5 0.05 3 82 5 4 0.06 4 81 7 4 0.09 5 80 8 4 0.10 6 78 9 30.12 7 77 10 3 0.13 8 76 11 3 0.15 9 75 13 2 0.17 10 74 14 2 0.19

[0091] The Example shows how protein precipitates can be obtainedaccording to the invention from a plasma protein mixture by a singleprecipitation with a mixture of two components (here, glycine and PEG),which protein precipitates contain variable amounts of fibronectin inaddition to the main component fibrinogen, wherein the ratio fibronectin: fibrinogen can be adjusted as desired within certain limits (e.g. byselection of the agents according to Example 1 in a range of between0.02 -0.2) by a suitable choice of the concentrations of the agents andtheir ratio to each other, respectively. Such protein precipitates are,e.g., suitable for producing fibrinogen-based tissue adhesives (fibrinadhesives).

Example 2

[0092] Human plasma cryoprecipitate was dissolved analogous to Example1, the pH was adjusted to 7.3, and it was filtered to clarification.Aliquots of this solution were admixed analogous to Example 1 withsolutions containing glycine and PEG 4000, so that in each case a finalPEG concentration of 6.5% (w/v), yet various glycine concentrationsranging from 0.2 to 1 M were obtained. The precipitates formed werecentrifuged off analogous to Example 1 and analyzed.

[0093] In all variants, the protein yield was approximately 95%. Theratio fibronectin : fibrinogen in dependence on the glycineconcentration has been summarized in Table 2 and is additionallygraphically represented in FIG. 2. TABLE 2 Dependence of the proteincomposition of the precipitate on the glycine content in theprecipitation mixture (PEG concentration: 6.5% w/v). GlycineFibronectin: mol/l Fibrinogen 0.2 0.16 0.4 0.16 0.6 0.16 0.8 0.14 1.00.11

[0094] Supplementing Example 1, this Example illustrates that proteinprecipitating agents known per se, such as glycine, by no means merelyact as precipitating agents when used in combination with PEG, but thatunder certain conditions glycine, e.g., may also act as a solubilizerfor fibronectin, this being in a range of conentration in which hithertoglycine was merely known as a protein-precipitating agent.

[0095] The following Examples 3-5 show comparable compositions ofpreparations obtained according to the prior art.

Example 3

[0096] A plasma cryoprecipitate was dissolved as in Example 1. Whilestirring at room temperature, glycine was added up to a finalconcentration of 2 mol/l. The protein precipitate formed was centrifugedoff and analyzed as in Example 1. Fibrinogen was almost completelyprecipitated. The relative content of fibrinogen amounted to 86% of thetotal protein, the relative content of fibronectin was 1.5%. The ratiofibronectin fibrinogen thus was 0.017.

Example 4

[0097] A plasma cryoprecipitate was dissolved as in Example 1. Whilestirring at room temperature, PEG 4000 was added up to a finalconcentration of 10%(w/v). The protein precipitate formed wascentrifuged off and analyzed as in Example 1. Fibrinogen was almostcompletely precipitated. The relative content of fibrinogen amounted to72% of the total protein, the relative content of fibronectin was 16%.The ratio fibronectin fibrinogen thus was 0.22.

Example 5

[0098] A plasma cryoprecipitate was dissolved as in Example 1. Whilestirring at room temperature, PEG 4000 was added up to a finalconcentration of 10% (w/v). The protein precipitate formed wascentrifuged off, dissolved again, and glycine was added to the solutionto a final concentration of 2 M, under stirring at room temperature. Theprotein precipitate formed was centrifuged off again and analyzed as inExample 1. The relative content of fibrinogen amounted to 93% of thetotal protein, the relative content of fibronectin was 1.5%. The ratiofibronectin fibrinogen thus was 0.016.

[0099] Examples 3-5 show that with the methods known in the prior art itis not possible to arrive, in one or also in several consecutive steps,at protein mixtures having a defined ratio of fibronectin : fibrinogen,e.g., when using a combination of glycine and PEG in the concentrationsused in Example 1 or 2, to arrive at a ratio in the range of from 0.02to 0.2.

Example 6

[0100] Production of a lyophilized preparation according to theinvention, with two independent virus inactivation steps:

[0101] Human plasma cryoprecipitate prepared according to methods knownper se was dissolved with the 4-fold amount of a buffer solution (LPl)containing 20 mM sodium citrate, 120 mM sodium chloride, 5 mM tranexamicacid (t-AMCHA) as well as 1200 IU heparin/l, and was filtered toclarification. Subsequently, a so-called solvent-detergent (SD)treatment was carried out to inactivate enveloped viruses possiblypresent. To this end, a mixture of Triton X-100, Tween 80(Polysorbat-80K) and tri-n-butylphosphate (TNBP) was admixed to givefinal concentrations of 1% , 0.3% and 0.3% (v/v), respectively. Afterstirring for 1 h at RT, it was again filtered to clarification. Thesolution was admixed with the equal volume of a solution containing 2 Mglycine and 13% (w/v) PEG 4000 (precipitating composition) understirring at room temperature, stirred for 30 min and centrifuged. Thesediment was comminuted and again dissolved in LPl containing 1% Tween80, and the precipitation was repeated in the same manner. To remove theSD and precipitating reagents, the sediment was then comminuted andtreated in the cold (0-2° C.) 2×with the 10-fold amount of a buffersolution containing 10 mM Na₃ citrate and 5 mM t-AMCHA in the presenceof small amounts of Tween 80. The washed sediment was then dissolved ina buffer solution containing 10 mM Na₃ citrate and 5 mM t-AMCHA. Afteradjusting the protein concentration to 40 g/l, the solution waslyophilzed as a whole. For further virus inactivation, the lyophilizedmaterial was adjusted to a residual moisture of 7-8% and heated for 10 hat 60° C.+1 h at 80° C. under the exclusion of oxygen. The thus-treatedlyophilisate was dissolved in 20 mM niacinamide solution to a proteinconcentration of 38 g/l, admixed with 6 g of pasteurized humanalbumin/l, and the pH was adjusted to 7.3

[0102] The solution was sterile-filtered, filled at 5.0 ml each intofinal containers (small glass bottles) under sterile conditions andlyophilized.

[0103] The final product thus obtained gave a ready-to-use solution whendissolved with 2.0 ml of water for injection (WFI), or with 2.0 ml of 50mM t-AMCHA solution, having a fibrinogen content of more than 70 mg/ml.This solution may, e.g., be used as a tissue adhesive.

[0104] In principle, instead of using pure water, the lyophilized finalproduct may also be dissolved with aqueous solutions containingadditional active substances, such as, e.g., fibrinolysis inhibitors,coagulation factor XIII, antibiotics, growth factors, pain-alleviatingsubstances etc.

[0105] The product produced according to the above-described method inits protein content and composition substantially corresponded to thetissue adhesive preparation described in EP 804933A2 (Example 2). Thefibronectin : fibrinogen ratio was approximately 0.09, the plasminogencontent merely about 0.15 mg/g of fibrinogen. After mixing theready-to-use tissue adhesive solution with the equal volume of athrombin-CaCl₂ solution, physiological, non-transparent, visco-elasticclots were formed.

[0106] Despite this broad agreement with the product described in EP804933, the tissue adhesive according to the invention is characterizedby a markedly reduced viscosity, with an identical content of asubstance improving the solubility of fibrinogen (50 mM niacinamide).

[0107] This is the more surprising as the preparation according to theinvention had not only been subjected to one, but had been subjected totwo independent virus inactivation steps and, moreover, the vaportreatment had been carried out under aggravated conditions (10 hours,60° C., +1 hour, 80° C.). According to experience, such a heat treatmentleads to a poorer solubility and an increased viscosity.

[0108] Therebeyond, surprisingly it has been found that t-AMCHA, inaddition to its known antifibrinolytic effect, lowers the viscosity offibrinogen-containing solutions. In particular, the viscosity of thetissue adhesive solutions obtained according to the invention is furtherlowered by t-AMCHA (cf. Table 3). TABLE 3 Viscosity [cSt] in thepresence of 50 mM niacinamide Tissue adhesive Tissue adhesive accordingto Temperature Tissue adhesive acc. to invention + EP 804933A2 [° C.]acc. to invention 50 mM t-AMCHA (Example 2) 20 99 78 277 25 76 63 132 3056 52 81 37 39 34 53

Example 7

[0109] Production of a liquid deep-frozen tissue adhesive according tothe invention with two independent virus inactivation steps:

[0110] Up to sterile filtration, the production was carried outanalogous to Example 6. Subsequently, the sterile-filtered solution waslyophilized under sterile conditions once more, dissolved to aconcentrated tissue adhesive solution, filled into final containers.(disposable syringes) and stored in the deep-frozen state. Prior to use,such a preparation need only be thawed.

[0111] The ready-to-use tissue adhesive solution had the same propertiesas the dissolved preparation of Example 6.

[0112] Instead of lyophilizing again the sterile filtered, dilutedtissue adhesive solution and to dissolve it in concentrated state, itmay also be evaporated directly under mild vacuum to a concentratedtissue adhesive solution.

[0113] Methodology of viscosity measurement

[0114] To standardize the measurement method, a sample of the tissueadhesive solution initially is frozen at ≦−20° C. To determine itsviscosity, the sample is thawed in a water bath at the desiredmeasurement temperature, incubated for approximately 30 min at thistemperature and then its viscosity is determined in atemperature-controlled capillary viscosimeter. Subsequently, the samplecan be incubated at a higher temperature in the viscosimeter, and themeasurement may be repeated at that temperature. The individualmeasurements are effected in series at rising temperatures. If measuredin the reverse sequence, falsified values (too low) could be obtained,since the equilibrium is reached only slowly at decreasing temperatures.

Example 8

[0115] A plasma cryoprecipitate was dissolved as in Example 1, the pHwas adjusted to 7.3, and it was filtered to clarification. Analogous toExample 1, aliquots of this solution were admixed with solutionscontaining β-alanine and ethanol, yet not at RT, but at 0° C. so that ineach case an ethanol concentration of 2% (v/v), yet different β-alanineconcentrations ranging from 0-1 M were obtained. The precipitates formedwere centrifuged off analogous to Example 1 and analyzed. In FIG. 3, theratio Fn/Fbg in dependence on the β-alanine concentration is graphicallyrepresented. TABLE 4 Dependence of the protein composition of theprecipitate on the β-alanine content in the precipitation mixture(ethanol concentration: 2%) β-Alanine [M] Fn/Fbg 0.0 0.43 0.2 0.34 0.40.30 0.6 0.26 0.8 0.25 1.0 0.23

[0116] Analogous to Example 2, this Example shows that proteinprecipitating agents known per se, such as β-alanine, when used incombination with ethanol, by no means merely act as a precipitatingagent, but that under certain conditions β-alanine, e.g., may also actas a solubilizer for fibronectin.

[0117] The following Examples 9-11 show comparable compositions ofpreparations obtained according to the prior art.

Example 9

[0118] A plasma cryoprecipitate was dissolved as in Example 1. Understirring at 0° C., β-alanine was added up to a final concentration of 2M. The protein precipitate formed was centrifuged off and analyzed as inExample 1. The relative content of fibrinogen amounted to 74% of thetotal protein, the relative content of fibronectin to 16%. The ratioFn/Fbg thus was 0.22.

Example 10

[0119] A plasma cryoprecipitate was dissolved as in Example 1. Understirring at 0° C., ethanol was added up to a final concentration of 2%(v/v). The protein precipitate formed was centrifuged off and analyzedas in Example 1. The relative content of fibrinogen amounted to 67% ofthe total protein, the relative content of fibronectin to 29%. The ratioFn/Fbg thus was 0.43.

Example 11

[0120] A plasma cryoprecipitate was dissolved as in Example 1. Understirring at 0° C., ethanol was added up to a final concentration of 2%(v/v) The protein precipitate formed was centrifuged off, dissolvedagain, and β-alanine was added to the solution under stirring at 0° C.up to a final concentration of 2 M. The protein precipitate formed wascentrifuged off again, and analyzed as in Example 1. The relativecontent of fibrinogen amounted to 77% of the total protein, the relativecontent of fibronectin to 16%. The ratio Fn/Fbg thus was 0.21.

[0121] Examples 9-11 show that with the methods known in the prior artit is not possible, in one or also in several consecutive steps, toarrive at protein mixtures having defined ratios of Fn/Fbg, e.g. whenusing a combination of β-alanine and ethanol at the concentrations usedin Exmaple 8, to arrive at a ratio ranging from 0.23-0.34.

Example 12

[0122] A plasma cryoprecipitate was dissolved as in Exmaple 1, the pHwas adjusted to 7.3, and it was filtered to clarification. Analogous toExample 1, aliquots of this solution were admixed with solutionscontaining β-alanine and ammonium sulfate, so that in each case aβ-alanine concentration of 1 M, yet different ammonium sulfateconcentrations ranging form 5-15% (w/v) were obtained. The precipitatesformed were centrifuged off analogous to Example 1 and analyzed. Theresults have been summarized in Table 5; in FIG. 4, the ratio Fn/Fbg independence on the ammonium sulfate concentration is graphicallyrepresented. TABLE 5 Dependence of the protein composition of theprecipitate on the ammonium sulfate content in the precipitation mixture(β-alanine concentration: 1 M) Ammonium sulfate Fbg Fn Alb % (w/v) (%Prot.) (% Prot.) (% Prot.) Fn/Fbg 5 69 5 0 0.07 10 78 12 0 0.15 15 78 160 0.21

[0123] Similar to Example 1, this Example shows how protein precipitatescan be obtained from a plasma protein mixture by a single precipitationwith a precipitating composition comprising two components (here,β-alanine and ammonium sulfate), which protein precipitates containvariable amounts of fibronectin, wherein the ratio Fn/Fbg can beadjusted as desired within certain limits (e.g. by selection of thecomponents according to Example 12 in a range of between 0.07 and 0.21)by a suitable choice of the concentrations of the components and theirratio to each other, respectively.

Example 13

[0124] A plasma cryoprecipitate was dissolved as in Example 1, the pHwas adjusted to 7.3, and it was filtered to clarification. Analogous toExample 1, aliquots of this solution were admixed with solutionscontaining β-alanine and ammonium sulfate, so that in each case anammonium sulfate concentration of 5% (w/v), yet different β-alanineconcentrations ranging from 0.6 - 1.4 M were obtained. The precipitatesformed were centrifuged off analogous to Example 1 and analyzed. In FIG.5, the ratio Fn/Fbg in dependence on the β-alanine concentration isgraphically represented. TABLE 6 Dependence of the protein compositionof the precipitate on the β-Ala content in the precipitation mixture(ammonium sulfate concentration: 5%) β-Alanine [M] Fn/Fbg 0.6 0.19 1.00.07 1.4 0.04

[0125] Analogous to Example 2, this Example shows that proteinprecipitating agents known per se, such as β-alanine, when used incombination with ammonium sulfate by no means merely act asprecipitating agents, but that under certain conditions β-alanine, e.g.,may also act as a solubilizer for fibronectin.

[0126] The following Examples 14-16 show comparable compositions ofpreparations obtained according to the prior art.

Example 14

[0127] A plasma cryoprecipitate was dissolved as in Example 1. Understirring at RT, β-alanine was added up to a final concentration of 2 M.The protein precipitate formed was centrifuged off and analyzed as inExample 1. The relative content of fibrinogen amounted to 82% of thetotal protein, the relative content of fibronectin to 1%. The ratioFn/Fbg thus was 0.01.

Example 15

[0128] A plasma cryoprecipitate was dissolved as in Example 1. Understirring at RT, ammonium sulfate was added up to a final concentrationof 15% (w/v) The protein precipitate formed was centrifuged off andanalyzed as in Example 1. The relative content of fibrinogen amounted to76%t of the total protein, the relative content of fibronectin to 16%.The ratio Fn/Fbg thus was 0.21.

Example 16

[0129] A plasma cryoprecipitate was dissolved as in Example 1. Understirring at RT, ammonium sulfate was added up to an ammonium sulfateconcentration of 5% (w/v). The protein precipitate formed wascentrifuged off, dissolved again, and β-alanine was added to thesolution under stirring at RT up to a final concentration of 2 M. Theprotein precipitate formed was centrifuged off again and analyzed as inExample 1. The relative content of fibrinogen amounted to 84% of thetotal protein, the relative content of fibronectin to 1%. The ratioFn/Fbg thus was 0.01.

[0130] Examples 14-16 show that with the methods known in the prior artit is not possible, in one or also in several consecutive steps, toarrive at protein mixtures having a defined ratio of Fn/Fbg, e.g. whenusing a combination of β-alanine and ammonium sulfate at theconcentrations used in Example 13, to arrive at a ratio ranging from0.04-0.19.

1. A method for producing a pharmaceutical preparation comprisingfibrinogen and firbronectin, characterized in that a fibrinogen andfibronectin-containing starting solution is treated with a precipitatingcomposition which comprises two different components that modify thesolubility of fibrinogen and/or fibronectin, so that in a single-stepprecipitation, a precipitate is formed which comprises fibrinogen andfibronectin.
 2. A method according to claim 1, characterized in that asolution derived from cryoprecipitate is used as the starting materialfor preparing the starting solution.
 3. A method according to claim 1,characterized in that the solubility-modifying component is selectedfrom the group consisting of alcohols having up to four carbon atoms,inorganic salts, organic salts, organic polymers, polyols, and aminoacids.
 4. A method according to claim 3, characterized in that thesolubility-modifying component is selected from the group consisting ofethanol, polyalkylene glycol, polyethylene glycol, ammonium sulfate,alkali sulfate, glycine and alanine.
 5. A method according to claim 4,characterized in that the precipitating composition comprisespolyalkylene glycol, and polyalkylene glycol is admixed to the startingsolution to a final concentration of from 1 to 12% w/v, preferably 4 to8% w/v.
 6. A method according to claim 3, characterized in that theprecipitating composition comprises one or more amino acids and isadmixed to the starting solution to a final concentration of from 0.5 to3 M, preferably 0.75 to 2 M, in particular 0.75 to 1.5 M.
 7. A methodaccording to claim 1, characterized in that the precipitatingcomposition is admixed in liquid form.
 8. A method according to claim 1,characterized in that a precipitating composition is used in whichplasminogen largely remains in the supernatant.
 9. A method according toclaim 1, characterized in that additional ingredients of the startingmaterial, in particular factor XIII, are precipitated by theprecipitating composition.
 10. A method according to claim 1,characterized in that the starting material and/or the starting solutionis checked for the absence of pathogens, in particular viruses and/orprions.
 11. A method according to claim 1, characterized in that atleast one step for inactivating or depleting possibly present pathogensis carried out before or after the precipitation.
 12. A method accordingto claim 11, characterized in that a heat treatment, a treatment with asolvent, a treatment with a detergent or a combination of thesetreatments is carried out for inactivation of possibly presentpathogens.
 13. A method according to claim 11, characterized in that thedepletion is carried out by means of filtration, in particularultrafiltration, preferably in the presence of virus-binding agents. 14.A method according to claim 1, characterized in that at least oneingredient selected from the group consisting of fibrinolysisinhibitors, factor XIII, antibiotics, growth factors, pain-alleviatingsubstances, and anti-inflammatory agents is admixed to the proteincomposition after the single-step precipitation.
 15. A method accordingto claim 1, characterized in that the pharmaceutical preparation isdeep-frozen or lyophilized in the course of further processing.
 16. Apharmaceutical preparation comprising fibrinogen and fibronectinobtained according to the method of claim
 1. 17. A pharmaceuticalpreparation according to claim 16, characterized in that it is presentin lyophilized, liquid, or in liquid-deep-frozen form.
 18. Apharmaceutical preparation according to claim 16, characterized in thatis has a ratio of fibronectin to fibrinogen in a range of from 0.02 to0.5.
 19. A pharmaceutical preparation according to claim 18 having aratio of fibronectin to fibrinogen in a range from 0.02 to 0.25.
 20. Apharmaceutical preparation according to claim 19 having a ratio offibronectin to fibrinogen in a range from 0.02 to 0.2.
 21. Apharmaceutical preparation according to claim 20 having a ratio offibronectin to fibrinogen in a range from 0.04 to 0.16.
 22. Apharmaceutical preparation according to claim 21 having a ratio offibronectin to fibrinogen of 0.1.
 23. A pharmaceutical preparationaccording to claim 16 further comprising plasminogen at a concentrationno greater than 1.6 mg plasminogen per g fibrinogen.
 24. Apharmaceutical preparation according to claim 23 containing plasminogenat a concentration less than 0.8 mg plasminogen per g fibrinogen.
 25. Apharmaceutical preparation according to claim 24 containing plasminogenat a concentration less than 0.3 mg plasminogen per g fibrinogen.
 26. Apharmaceutical preparation according to claim 16 further comprising anactive substance selected from the group consisting of antibiotics,growth factors, and pain-alleviating substances.
 27. A pharmaceuticalpreparation according to claim 16, characterized in that a) as asolution, it contains at least 70 mg of fibrinogen/ml or can bereconstituted or liquefied, respectively, to such a solution, b) at 20°C., it has a viscosity of 350 cSt at the most, preferably at anosmolarity of less than 500 mOsm, most preferred of less than 400 mOsm,c)after mixing with a thrombin-CaCl₂ solution, it forms anon-transparent clot of physiological fibrin structure, and d)it doesnot contain an addition of a solubility-improving substance with abenzene, pyridine, piperidine, pyrimidine, morpholine, pyrrole,imidazole, pyrazole, furan, thiazole or purin-containing group.
 28. Apharmaceutical preparation according to claim 16, characterized in thata) as a solution it contains at least 70 mg of fibrinogen/ml. or can bereconstituted or liquefied, respectively, to such a solution, b) at 20°C., it has a viscosity of 150 cSt at the most, preferably at anosmolarity of less than 500 mOsm, most preferred of less than 400 mOsm,c) after mixing with a thrombin-CaCl₂ solution, it forms anon-transparent clot of physiological fibrin structure, and d) itcontains one or several solubilizing substances with a benzene,pyridine, piperidine, pyrimidine, morpholine, pyrrole, imidazole,pyrazole, furan, thiazole or purine-containing group at a concentrationof a total of 150 mM at the most.
 29. The use of a pharmaceuticalpreparation according to claim 16 for tissue adhesion, haemostatis, orwound healing.
 30. The use of a pharmaceutical preparation according toclaim 16 for producing a fibrin-based biomatrix.
 31. The use accordingto claim 30, characterized in that the biomatrix is formed as a film, anon-woven fabric or a sponge.
 32. The use according to claim 30,characterized in that the biomatrix is suitable for growing cells, inparticular human cells. 33.The use of the biomatrix according to claim30 for dressing wounds or as a tissue substitute, in particular as askin substitute.
 34. A biomatrix obtained from a pharmaceuticalpreparation according to claim 16.